GDC 0941

Mouse His₆-p110β(1-1064)/p85β-icSH2(423-722) complex was diluted to 4 mg/ml, mixed with 20 mM (final concentration) sodium phenyl phosphate (Sigma P-7751) and 150 μM of the PI3K inhibitor GDC0941 (Folkes et al., 2008 (link)). The initial crystallization conditions were obtained from a broad screen of 1056 conditions (Stock et al., 2005 (link)) in 96-well MRC crystallization plates (SWISSCI AG, Zug, Switzerland). Additives (GDC0941 and phenyl phosphate) were identified by differential scanning fluorimetry (see Supplemental Experimental Procedures). Optimal crystals were obtained at 22°C in hanging drops over reservoirs of 24-well plates (Hampton Research, Aliso Viejo, CA) containing 12% polyethylene glycol 3350, 0.1 M potassium citrate at pH 6, and 0.4 M lithium sulfate. The drops contained 1 μl each of protein and reservoir solutions. The crystals were cryoprotected by stepwise addition of cryoprotectants consisting of the reservoir solution with 20 mM sodium phenyl phosphate, 150 μM of GDC0941, and an increasing concentration of glycerol up to 20% (in 5% increments). Crystals were flash frozen in liquid nitrogen.

Fresh Her2+ and TN human tumor samples were acquired from patients undergoing resection of breast to brain metastases, in accordance with a City of Hope Institutional Review Board (IRB)-approved protocol (IRB #05091). A portion of each specimen was cultured in DMEM-F12 (Life Technologies) supplemented with 10% fetal bovine serum (FBS), 1% glutamax, and 1% Anti-Anti (Life Technologies) in collagen-coated (Life Technologies) T75 flasks to derive low-passage primary cell lines (COH-BBM1 (BBM1), and COH-BBM2 (BBM2) and COH-BBM3 (BBM3)) [19 (link)]. Established breast cancer cell lines MDA-MB-361 (361), BT474 and SkBr3 cells were also cultured in the aforementioned DMEM-F12 in T75 flasks. All cells were maintained at 37 °C and 5% CO₂.
To obtain conditioned medium from astrocytes and fibroblasts, cells were grown in serum-free DMEM for 24 h before collecting and purifying the medium by centrifugation (4000 × g, 15 minutes). For growth in conditioned medium, BBM cells were first grown for 24 h in serum-free DMEM. The growth medium was removed and the cells were washed once with fresh DMEM before adding the conditioned medium. Cells were grown for differing amounts of time in the conditioned medium, as indicated for specific experiments. For treatment with BDNF, lapatinib, cyclotraxin B, XL 147 or GDC0941, cells were grown overnight in serum-free mediuma before treatment with BDNF, lapatinib and cyclotraxin B for various time periods. For details on materials and methods please see Additional file 1.

LDH Cytotoxicity assay was performed using Pierce LDH cytotoxicity assay kit (Thermo scientific 88,954) according to manufacturer’s instructions. Briefly, the optimal number of cells (1 × 10⁵ cells/ml) were seeded in 100 μl of RPMI medium in triplicate wells in a 96-well tissue culture plate. A complete medium control without cells was used to determine LDH background activity present in sera used for media supplementation. A serum-free media control was included to determine the amount of LDH activity in sera. Additional cells were plated in triplicate wells for spontaneous LDH activity controls (water) and maximum LDH activity controls (10X Lysis Buffer). The plate was incubated in an incubator at 37°C, 5% CO₂ for 30 min. To the set of triplicate wells serving as the maximum LDH activity controls, 10 μl of lysis buffer (10X) was added, and mixed by gentle tapping. Different concentrations of glucose (5 mM and 15 mM), and inhibitors AZD6244 (10 μM), GDC0941 (2 μM), GDC0068 (2 μM), were added to cells. The plate was again incubated in an incubator at 37°C, 5% CO₂ for 45 min. Each sample medium (50 μl) (e.g., complete medium, serum-free medium, Spontaneous LDH Activity Controls, glucose-treated, inhibitors-treated and Maximum LDH Activity Controls) was added to a 96-well flat-bottom plate in triplicate wells. Reaction mixture (50 μl) was transferred to each sample well and mixed using a multichannel pipette. The plate was incubated at room temperature for 30 min protected from light. Stop solution (50 μl) was added to each sample well and mixed by gentle tapping. The absorbance was measured at 490 nm and 680 nm. To determine LDH activity, the 680 nm absorbance value (background) was subtracted from the 490 nm absorbance before calculation of % cytotoxicity [(LDH at 490 nm) − (LDH at 680 nm)]. The % cytotoxicity was calculated as follows:
% Cytotoxicity = Glucose/inhibitors-treated LDH activity − Spontaneous LDH activity/Maximum LDH activity − Spontaneous LDH activity × 100.

The synergy of drug combinations was evaluated in the mouse 5746 and TM31 glioma cell lines. The 17 glioma cell line was used for the PTC596/Trametinib combination as this combination showed limited sensitivity than the 5746 line (S2 Table). Cell lines were plated in 96-well plates and processed as described in the spheroid or 2D drug screening assays. We created a dilution series of our drug combinations (7 dilutions of the drug of interest, 5 dilutions of Trametinib or GDC-0941). On Day 0, Trametinib, GDC-0941, Bortezomib, Dinaciclib, JQ1, LY2606368, PTC596, or Vorinostat were added in a serial dilution per row in duplicate. We used the percent viability compared to untreated cells to calculate a synergy score for each of the analyzed combinations (https://synergyfinder.fimm.fi/; parameters: LL4 curve fit, ZIP method for synergy score calculation).